Chapter Introduction
Table of Contents Introduction Page. Principles of PCR and RT-PCR...9.2 The Evolution of PCR....3 Purpose of this PCR Applications Manual...5 8 PCR Applications Manual
Principles of PCR and RT-PCR. Introduction. Principles of PCR and RT-PCR If DNA is the master blueprint of life, DNA polymerase is the molecule most responsible for replication and dissemination of the blueprint. Without DNA polymerase, most living organisms could not generate future progeny or evolve. The principal function of DNA polymerase is to synthesize new strands of DNA in a 5-3 direction from a single-stranded template. Most native DNA polymerases, however, are polyfunctional. In addition to their strand synthesis activity, many DNA polymerases are able to remove nucleotides sequentially from either end of the strand (5 exonuclease and/or 3 exonuclease activities). Molecular biologists have based an incredible number of laboratory applications on the multiple activities of DNA polymerase. In April 983, Kary Mullis of Cetus Corporation took a drive on a moonlit California road and came up with the simplest, most elegant application of all. During that drive, he conceived the basic idea for the Polymerase Chain Reaction (PCR), which is a technique for amplifying a specific target DNA sequence in vitro. Ten years later, Dr. Mullis received the 993 Nobel Prize in Chemistry, underscoring the importance of that one simple idea. The PCR Cycle PCR amplification can turn a few molecules of a specific target nucleic acid (too little to be analyzed directly or used in biochemical reactions) into as much as a microgram of DNA. PCR is closely patterned after the natural DNA replication process (Saiki et al., 985). Two oligonucleotide primers flank and define the target sequence to be amplified. These primers hybridize to opposite strands of the DNA to serve as initiation points for the synthesis of new DNA strands. A thermostable DNA polymerase, such as Taq DNA Polymerase, catalyzes this synthesis. Each round of PCR synthesis involves three steps: denaturation, annealing and extension. This three-step PCR cycle is repeated (Figure.) several times (Mullis and Faloona, 987). The repetitive nature of the PCR process is the key to its amplifying power. Because the primer extension products synthesized in a given cycle can serve as templates in the next cycle, the number of target DNA copies approximately doubles every cycle. Thus, in only 20 cycles, PCR can produce about a million (2 20 ) copies of the target. RT-PCR In 987, Powell et al. described a technique that extended the power of PCR to the amplification of RNA. This technique, RT-PCR, used a reverse transcriptase to convert the rare RNA into a cdna, then used a thermostable DNA polymerase to amplify the cdna to detectable levels. This technique made it possibe to use PCR to detect and analyze rare mrna transcripts and other RNAs present in low abundance. For more information on RT-PCR, see Chapter 5 in this manual. Introduction 9
Principles of PCR and RT-PCR PCR Step : Denaturation of template with heat PCR Step 2: Annealing of primers to template PCR Step 3: Extension of primers with thermostable polymerase Step : Heat (usually >90 C) separates double-stranded DNA into two single strands. Since the hydrogen bonds linking the bases to one another are weak, they break at high temperatures, whereas the bonds between deoxyribose and phosphates, which are stronger covalent bonds, remain intact. This process takes place in a thermal cycler, an instrument that automatically controls the alternating cycles of heating and cooling required for PCR. Step 2: The goal is not to replicate the entire strand of DNA but to replicate a target sequence of approximately 00-35,000 base pairs that is unique to the organism. Primers define the ends of that target sequence. Primers are short, synthetic sequences of single-stranded DNA typically consisting of 20-30 bases. [Theoretically, a 6-mer is long enough to represent all unique primer sequences (4 6 ) in a random sequence of 3 billion base pairs.] Annealing usually takes place between 40 C and 65 C, depending on the length and sequence of the primers. This allows the primers to anneal specifically to the target sequence. Step 3: Once the primers anneal to the complementary DNA sequences, the temperature is raised to approximately 72 C and a thermostable polymerase (e.g., Taq DNA Polymerase) begins to synthesize new double-stranded DNA molecules which are identical to the original target DNA. It does this by facilitating the binding and joining of complementary nucleotides that are free in solution (dntps). Synthesis always begins at the 3' end of the primer and proceeds exclusively in the 5' to 3' direction. Thus, the new synthesis effectively extends the primers, creating a complementary, doublestranded molecule from a single-stranded template. End of the first PCR cycle: Two copies of target sequence End of cycle: At the end of the first PCR cycle, there are now two new DNA strands identical to the original target. Note, however, that the DNA polymerase does not recognize the end of the target sequence. The newly formed strands have a beginning, which is precisely defined by the 5' end of the primer, but not a precisely defined 3' end. Each subsequent cycle repeats and multiplies this copying process. However, as the number of cycles increases, a strand with more defined length frequently serves as the template for the newly synthesized sequence. The DNA strand synthesized from such a template then has a precisely defined length that is limited at either end by the 5' end of each of the two primers. These DNA strands are called an amplicon. After only a few cycles, DNA strands which correspond to the target sequence are present in much larger numbers than the variable length sequences. In other words the sequence flanked or defined by the two primers is the section that is amplified. After the appropriate number of PCR cycles (usually between 30 and 40 cycles), this repetitive, sequential process exponentially generates up to a billion of copies of the target, all within just a few hours. Figure.. The PCR Cycle. PCR is a cyclic DNA amplification process. Each cycle involves three steps, which are described in detail above. When the cycle is repeated several times, the net result is a rapid increase in the total number of copies of the target DNA. 0 PCR Applications Manual
The Evolution of PCR.2 The Evolution of PCR The earliest version of PCR was certainly revolutionary and important, but it had some serious limitations (McPherson and Møller, 2000). Initially, the DNA was amplified with the Klenow Fragment of DNA pdna polymeraseolymerase I. However, that enzyme was not stable at high temperatures. Thus, the Klenow in the tube was inactivated during the denaturation step of each PCR cycle, and the experimenter had to add more Klenow before each extension step. Morever, the extension had to be performed at a relatively low temperature (37 C). That made the technique boring for the scientist (because addition of Klenow was monotonous and repetitive), expensive (because a large amount of Klenow was required) and likely to introduce nonspecific products (since the primers could bind to nontarget regions of DNA at 37 C, resulting in amplification of these regions). The technique was also susceptible to outside contamination (since the tube had to be opened repeatedly to add new enzyme). Lastly, scientists could only handle a few samples at a time and, since they had to be present throughout the reaction, could not easily automate the PCR process. Clearly, scientists solved all those limitations over time to develop the modern version of PCR, which has become an indispensible tool in almost every laboratory. The evolution of the PCR technique is mainly a story of enzymes, equipment and enhanced applications. Roche Applied Science (RAS) as well as other divisions, have made key contributions to the story in each of these areas. The following sections provide a brief overview of some of these contributions. PCR Enzymes The potential of the PCR technique increased dramatically when scientists identified a thermostable DNA polymerase, Taq DNA Polymerase, that was optimally active and stable at the high elongation temperature (around 72 C) used in PCR. Because the Taq enzyme was stable during the repeated PCR cycles, researchers no longer had to interrupt the PCR process to add fresh enzyme (Saiki et al., 988). Introduction
The Evolution of PCR 989 The native Taq enzyme was purified in 986 by David Gelfand and Susanne Stoffel (who were then employed by Cetus Corporation, but later assumed important roles at Roche Molecular Diagnostics). Boehringer Mannheim (now Roche Applied Science) was one of the companies that provided a convenient recombinant version of this important enzyme (in 989). Taq DNA polymerase still had drawbacks however. One such drawback was that it lacked a proofreading activity that could correct the occasional transcriptional errors (and potential mutations) that occurred during amplification. For many applications, these occasional mutations were of little concern. However, in some applications (e.g., the amplification of genomic products for sequencing and the study of allelic polymorphisms), any transcriptional errors can lead to misleading results. The commercial availability of thermostable enzymes that had proofreading activity solved this problem, providing a high fidelity version of PCR for those applications that required very accurate transcription. 994 A thermostable, proofreading enzyme, Pwo DNA Polymerase, was introduced by Boehringer Mannheim (later Roche Applied Science) in 994. Another approach to solving the accuracy problem was to combine Taq DNA polymerase with a thermostable enzyme (such as Tgo DNA polymerase) or other protein that had proofreading activity. 995 One such enzyme blend, the Expand High Fidelity PCR System, was introduced by Boehringer Mannheim (later Roche Applied Science) in 995. Replacing a single enzyme with an enzyme blend also led to another important advance in the evolution of PCR, the ability to amplify much longer targets (Barnes, 994). 996 By skillfully purifying and blending enzymes, then pairing the blend with carefully optimized reaction components, Roche Applied Science was able to offer one enzyme blend (Expand Long Template PCR System, introduced in 994) that could amplify up to 20 kb targets and another (Expand 20 kb PLUS PCR System, introduced in 996) that could amplify up to 35 kb targets. By modifying Taq DNA polymerase so it was inactive at room temperature, but readily activated at DNA denaturing temperatures, researchers made possible a hot start version of PCR that minimized the formation of troublesome primer dimers (Birch et al, 996). 2000 Roche Applied Science introduced (in 2000) FastStart Taq DNA Polymerase for hot start applications. By including FastStart Taq DNA Polymerase in an enzyme blend (FastStart High Fidelity PCR System, introduced in 2003), Roche Applied Science created a high fidelity hot start PCR system that can be used in such demanding applications as multiplex PCR. 2 PCR Applications Manual
The Evolution of PCR PCR Equipment In early PCR experiments, researchers had to rely on a series of water baths to maintain the different temperatures required by the procedure. Cycling involved manual transfer of samples from one water bath to another at specified times. In 988, Perkin-Elmer introduced the thermal block cycler, a revolutionary device that automatically and repetitively raised and lowered the temperature of the samples during the PCR cycles. This allowed the PCR technique to be automated. Subsequent refinements of this device extended the flexibility and accuracy of PCR. In 99, Holland et al. described a technique (5 nuclease assay) to simultaneously amplify and detect specific DNA sequences with a fluorescent DNA-binding dye. This technique (a 5' nuclease assay) uses so called hydrolysis probes and similar FRET (fluorescence resonance energy transfer)-based techniques made it possible to analyze PCR products as they were being formed (so-called kinetic or real-time PCR analysis). For more information on real-time PCR, see Chapter 7 in this manual. In 997, Boehringer Mannheim (later Roche Applied Science) licensed the LightCycler Instrument technology from Idaho Technology to take advantage of these real-time PCR techniques. By using smaller samples and improved analytical software, the LightCycler Instrument greatly reduced the time required for PCR amplification and identification of products. Eventually, real-time PCR devices became sophisticated enough to rapidly quantify specific DNA sequences in multiple samples. This allows laboratories to use PCR for high-throughput screening (e.g., for gene expression or genetic variation analyses) or to develop diagnostic PCR tests (e.g., for clinical or forensic purposes). Roche Applied Science is a major supplier of reagents and instrumentation for such research. The latest such Roche Applied Science instrument, the LightCycler 480 Real-Time PCR System (introduced in 2005), can automatically analyze up to 384 samples in a single run. Introduction 3
The Evolution of PCR PCR Applications Because of PCR, insufficient nucleic acid is no longer a limitation in research and many medical diagnostic procedures. Most importantly, innovative researchers have continually updated and expanded the definition of PCR applications, increasing the usefulness and scope of the technique. Even a brief review of these innovative applications is beyond the scope of this article. However, here is just a random sampling of PCR applications that are currently important: Automated PCR instruments that permit real-time detection and analysis of many products in a single run (e.g., the LightCycler Carousel-based System, the LightCycler 480 Real-Time PCR System, and the COBAS TaqMan 48 Analyzer). Clonal amplification of genomic DNA in an emulsion (empcr) to make ultrarapid sequencing of the genome possible (e.g., in the Genome Sequencer 20 System). Introduction of molecular tags (such as biotin and digoxigenin) into the PCR product during amplification, allowing these products to be used as sensitive hybridization probes in medical diagnostic tests (e.g., in the COBAS AMPLICOR Analyzers). Simultaneous amplification of multiple sequences in a single sample (multiplex PCR) (e.g., for identification of human single nucleotide polymorphisms in genomic DNA). Studies of genetic variability (e.g., to determine the genetic basis of diseases). Amplification of DNA for identity testing (e.g., DNA fingerprinting). Study of epigenetic mechanisms (such as DNA methylation, histone acetylation, and RNA interference) involved in activation and inactivation of genes (e.g., addition of T7 promoters to DNA templates to allow generation of double-stranded RNA for gene knockdown studies). Creation of novel DNAs by in vitro mutagenesis. Exploration of evolutionary relationships via examination of ancient DNA from fossils. Each of these applications have led to many more published articles than can be mentioned here. 4 PCR Applications Manual
Purpose of this PCR Applications Manual.3 Purpose of this PCR Applications Manual PCR has come a long way, but we still feel there is a need for a basic PCR handbook such as this PCR Applications Manual. This 3 rd edition of the manual repeats, extends and updates information contained in the previous two editions, including: General guidelines for setting up a PCR lab (chapter 2). Suggestions for optimizing the production of primers and templates (chapter 3). A basic set of PCR (chapter 4) and RT-PCR (chapter 5) protocols, including tips on how to get the best results with our products. Suggestions for cleaning up, analyzing and cloning PCR products (chapter 6). Guidelines to help you decide what PCR technique or reagent to use (protocol and product guides in chapters 3-6 and the Appendix). A brief look at some of the current applications for PCR and RT-PCR, which take advantage of products from Roche Applied Science (chapter 7). Applications from papers submitted to Roche Applied Science, each describing an application for one or more of the PCR and RT-PCR products described in previous chapters (chapter 8). A PCR toolbox that summarizes a wealth of technical information, including troubleshooting tips, useful PCR parameters, and detailed ordering information for all the products mentioned in this manual (Appendix). We hope that this information will be useful to both those new to PCR and those with a great deal of experience with the technique. Introduction 5
6 PCR Applications Manual